SUMMARY Toxoplasma gondii is an opportunistic protozoan parasite that poses a significant risk to AIDS/HIV patients. The currently available drugs for toxoplasmosis have significant adverse effects and are ineffective to eradicate long- lived tissue cysts located in the brain and muscles. Identifying new drug targets is imperative to control Toxoplasma infections with new drugs. Natural infection by T. gondii occurs via oral ingestion of tissue cysts (containing bradyzoites) or environmental oocysts (containing sporozoites). After ingestion of tissue cysts, excysted bradyzoites invade enterocytes where they transform into fast-replicating tachyzoite forms. After several cycles of replication in the small intestine and gut organs, tachyzoites migrate to the lamina propria where they enter the bloodstream to reach the brain and muscles to form tissue cysts. How Toxoplasma cysts are able to survive in the harsh environment of the stomach and gut tissues, and escape destruction by immune cells in the lamina propria remains largely unknown. Coccidian parasites (e.g., Toxoplasma), which need to be ingested orally by a host to initiate an infection, have in common the presence of genes coding for serine protease inhibitors that may target host serine proteases. Our proposal focuses on the contribution of a highly abundant serine protease inhibitor of T. gondii, named TgPI-1 to the protection of the parasite against host serine proteases present in the gut lumen (pancreatic elastase, trypsin, chymotrypsin), and/or secreted by immune cells in the lamina propria (neutrophil elastase, cathepsin G). We showed that TgPI-1 is secreted by tachyzoites and bradyzoites from dense granules, and inhibits trypsin, chymotrypsin and neutrophil elastase in vitro. Compared to wild-type parasites, TgPI-1- deficient parasites (cystogenic, type II strain) are more vulnerable to serine proteases added to the medium and have reduced dissemination in the gastro-intestinal tract of mice after oral administration of tissue cysts. Our hypothesis is that TgPI-1 contributes to the protection of T. gondii at the onset of infection in the gut. Specific Aim 1 will identify the TgPI-1 targets and sites where ?TgPI-1 parasites are killed in the gastro-intestinal tract to reveal the sites where TgPI-1 is secreted to protect Toxoplasma in the gut. Specific Aim 2 will focus on the contribution of TgPI-1 to protect the parasite in the lamina propria by inhibiting neutrophil elastase secreted by activated neutrophils or released by dying neutrophils during extrusion of Neutrophil Extracellular Traps (NET) wherein neutrophil elastase is highly abundant. From a therapeutic point-of-view, TgPI-1 could be a newly identified virulence factor, and its pharmacological inhibition in combination with current drug treatments, may increase the efficacy of anti-Toxoplasma treatments.